The objective of this design-driven proposal is to optimize and employ scaffold-free co-cultures for the generation of biomimetic fibrocartilages for the repair or replacement of the temporomandibular joint meniscus and the mandibular cartilage surfaces. Using co-cultures of fibrochondrocytes and articular chondrocytes, we recently generated large constructs of clinically relevant dimensions that were fibrocartilage-like in appearance and composed of extracellular matrix (ECM) suggestive of fibrocartilage. Moreover, constructs similar to native tissue were achieved with fibrochondrocytes. Motivated by these findings, it is our hypothesis that the co- cultures can be optimized using bioactive agents and mechanical stimuli to form biomimetic fibrocartilage constructs. To address this hypothesis, we propose the following specific aims: 1) to optimize the use of chondroitinase ABC and the growth factors transforming growth factor -1 (TGF-1) and insulin-like growth factor 1 (IGF-I) using serum-containing medium or chemically defined medium in the scaffold-free co-culture of fibrocartilage;2) to enhance the biomimetic fibrocartilage constructs with mechanical stimulation;and 3) to examine synergistic effects of bioactive agents and mechanical stimuli. Chondroitinase ABC has been found in preliminary studies to significantly increase construct tensile properties. Our group has also demonstrated that TGF-1 and IGF-I significantly increase ECM production in fibrocartilage constructs. The use of hydrostatic pressure and direct compression stimulation has been shown by our laboratory to have beneficial effects on articular cartilage and fibrocartilage constructs, and we will apply these stimuli individually and in combination to further enhance the constructs. Constructs will be examined histologically for glycosaminoglycan (GAG) and collagen, and immunohistochemically for collagen I and II. GAG, collagen, and DNA content will be quantified, followed by ELISA to measure collagen I and II. Biomechanical evaluation will include compression, tension, and creep indentation testing. Furthermore, microarray analysis will be used to study potential synergisms that may arise in the use of the exogenous stimuli. Finally, the engineered constructs will be implanted in the nude mouse to examine viability and stability. PUBLIC HEALTH RELEVANCE: Successful generation of biomimetic fibrocartilage would be a great stride toward the treatment of injuries or degeneration of the TMJ meniscus and mandibular cartilage surfaces, but much needs to be accomplished before this goal can be reached. The compositional and mechanical property heterogeneity of native fibrocartilages, further mirrored in their varying functions, necessitates development of a range of tissues for regeneration. In the course of identifying the optimal chondroitinase ABC, growth factor, and mechanical stimulation treatments, one for each of three cell ratio groups, this proposal will yield insight into the process of generating a spectrum of fibrocartilages with varying mechanical and compositional properties, biomimetically recreating the range of properties seen in the TMJ fibrocartilages. Furthermore, microarray analysis will elucidate the roles of these exogenous stimuli. The viability and stability of the engineered constructs will also be investigated in an immunodeficient animal model. The enabling technologies generated by this proposal can then be applied not only to tissue engineering one particular region of the TMJ meniscus or mandibular cartilage, but also to other fibrocartilaginous systems (e.g., knee meniscus) as well.